Specimen transport mount for spectrophotometer

ABSTRACT

SPECTROPHOTOMETRIC ANALYSES OF SPECIMENS WHICH HAVE BEEN PROCESSED BY &#34;DENSITY GRADIENT CENTRIFUGATION&#34; CAN BE PERFORMED BY THE APPARATUS DISCLOSED HEREIN WITHOUT HAVING TO WITHDRAW FRACTIONS OF THE MATERIAL FROM THE SPECIMEN TUBES FOR SEPARATE ANALYSIS. THE APPARATUS CONSTITUTES AN ATTACHMENT FOR A CONVENTIONAL SPECTROPHOTOMETER AND TRANSPORTS THE SPECIMENS IN A SPECIMEN TUBE THROUGH THE SPECTROPHOTOMETER&#39;&#39;S BEAM OF LIGHT IN A CONTROLLED SCANNING MOVEMENT PARALLEL TO AN ESTABLISHED DENSITY GRADIENT IN THE SPECIMEN.

March 2, 1971 v. T. RILEY SPECIMEN "iRANSPORT MOUNT FORSPECTROPHOTOMETER Filed June 2. 1966 2 Sheets-Sheet 1 no. 2.

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Film- 7 LOAD March 2,1971 v. T. RILEY 3,567,328

SPECIMEN TRANSPORT MOUNT FOR SPECTROPHOTOMETER Filed June 2. 1966 2Sheets-Shet FIG. '7.

INVENTOR.

3,567,328 SPECIMEN TRANSPORT MOUNT FOR SPECTROPHOTOMETER Vernon T.Riley, 2 South Road, Harrison, N.Y.

Filed June 2, 1966, Ser. No. 554,909 Int. Cl. G01 3/42; Gllln 1/00 US.Cl. 356-244 6 Claims ABSTRACT OF THE DISCLOSURE This invention relatesto a method and apparatus for the measurement of the optical density ofa specimen which has been subjected to a centrifugal field in anultracentrifuge and especially to such a specimen when it has beenprocessed in an ultracentrifuge by the method known as density gradientcentrifugation. This method produces bands of specimen material alongthe length of a specimen tube. The specimen may be placed in a preformeddensity gradient material, such as a sucrose varying in density from thebottom to the top of a centrifuge specimen tube. Upon being subjected tothe action of an ultracentrifuge, the specimen to be examined then seeksthe level of density which matches its own molecular density. If itconsists of a plurality of molecular species, each having a differentdensity, it is divided into a series of bands located at thecorresponding density region in the host liquid. The details of onemethod of this general type is described in a review by Howard Schachmanin Biochemistry, vol. 2, p. 887, Sept. 20, 1963.

It has been prior practice to withdraw fractions of the material fromthe tube, either at various locations of interest or at one end layer bylayer, slowly into a series of sample cuvettes and examine samephotometrically in a spectrophotometer or other photometric device, orin other ways. Correlating the samples examined with the appearance ofoptical absorption at a particular wavelength, it was possible toestablish the position or the centrifugal radius of the various zonesinto which the specimen had been decomposed by the centrifugation. Ithas been particularly advantageous to examine such specimens byspectrophotometry since the component substances can in this way bemeasured 'both qualitatively and quantitatively in thespectrophotometer. The optical absorption properties of substances, asis well known, are related to their molecular composition.

Although such examination by density gradient centrifugation has provento be a useful research tool, I have found marked advantages for a newsystem, using new equipment, in which the centrifuged specimen, in thehost liquid, is investigated in the specimen tube, without any need forit to be disturbed and placed into a series of sample cuvettes andwherein a continuous curve of optical density at a given wavelength maybe taken along the length of the specimen tube. By making continuousmeasurements in situ, many advantages are produced, some of which are asfollows: the material in each band cannot be contaminated because it isnot disturbed, the specimen can be re-examined often at variouswavelengths and the United States Patent ice curves compared, theinvestigator cannot make the mistake of missing a possibly importantband by selecting examining points above and below it, the materials canbe examined in undiluted form, one may easily take measurements of agiven sample at various times to produce a full record of migration withsize to obtain information as to molecular size, and one can easilyobtain information as to an infinite number of fractions over the fullrange of densities and with several wavelengths of lights. By using themethod and apparatus of the present invention, I have found things whichif they could have been found at all by prior techniques, would havetaken days rather than hours.

Accordingly, it is an object of the present invention to provide animproved method and apparatus for observing a sample which has beentreated in an ultracentrifuge. More specifically, it is an object ofthis invention to provide an attachment for a spectrophotometer whichwill transport a specimen through the spectrophotometers beam of lightin a controlled scanning movement parallel to an established densitygradient in the specimen.

It is further within the contemplation of the present invention toprovide an automatic transport mechanism for a liquid specimen for usein conjunction with a spectrophotometer which mechanism moves a specimenacross the beam path of a spectrophotometer such that the opticaldensity or light transmission of the specimen may be recorded as afunction of the position in the specimen at which the beam of lighttraverses it.

In accordance with one presently preferred illustrative embodiment ofthe invention, there is provided a specimen mount for use with aspectrophotometer of the type having a beam of light along a beam path.The specimen mount comprises a frame on which a specimen carriage ismounted by means of appropriate guide means for movement along acontrolled vertical path. The specimen carriage has means to secure thespecimen in a vertical orientation and is positioned such that thespectrophotometer beam path sweeps along the vertical extent of thespecimen as the carriage is moved along its vertical path. Drive meansare provided in the device for moving the carriage through a controlledvertical scanning movement to sweep the height of the specimen holderpast the beam. The specimen carriage may also be moved at a fasterrateto a loading location spaced from the beam path to provide ready accessto the carriage for the loading and unloading of specimens. Switchingmeans may be provided for automatically governing the drive means andfor denoting the beginning and end of the scanning movement. Theimproved specimen mount is associated with a recording device to recordthe signal from the spectrophotometer as a function of movement of thespecimen along its scanning path thereby to produce a graph of theoptical density of the specimen as it is measured against the relativevertical position of the specimen at which the optical density isrecorded.

The above brief description, as well as further objects, features andadvantages of the present invention, will be best appreciated by aconsideration of the following detailed description of one illustrativeembodiment of the invention when taken in conjunction with theaccompanying drawings wherein:

FIG. 1 is an elevational view of the attachment in accordance with thepresent invention shown positioned on a spectrophotometer with a portionof the device shown in phantom. This figure indicates the location ofthe beam path of the spectrophotometer;

FIG. 2 is an elevational view of the transport device in accordance withthe present invention, with most of the covering portions broken awayfor clarity, and shown mounted in a spectrophotometer and illustratingthe position of its various elements with respect to each other and withrespect to the spectrophotometer beam path;

FIG. 3 is a partial side sectional view taken at right angles to theview shown in FIG. 2 and taken along the line 3-3 of FIG. 2 and lookingin the direction of the arrows;

FIG. 4 is a sectional view taken along the line 44 of FIG. 2 and lookingdownwardly in the direction of the arrows;

FIG. 5 is a schematic diagram of the electrical switching and drivingmeans for the device in accordance with the present invention andillustrating the manner in which electrical interconnection is made tothe spectrophotometer;

FIG. 6 is a diagrammatic showing of the relative Loading and Readypositions of a specimen in the transport device in accordance with thepresent invention and comprises a diagrammatic showing of the variousmovements exhibited by a sample positioned in the device; and

FIG. 7 is an illustrative graph of the type produced by aspectrophotometer when using the transport mechanism in accordance withthe present invention with a diagrammatic showing of a specimen tubepositioned parallel to the horizontal axis of the graph for comparativepurposes.

Referring now to the drawings, there is shown in FIG. 1 an elevationalview of a portion of a photometer 10, specifically, a spectrophotometer10, with the location of its light beam diagrammatically indicated bythe arrow 12. A specimen mounting and transport device, generallydesignated by the numeral 14 and constructed in accordance with thepresent invention, is positioned on the spectrophotometer 10 with itsdownward extension 16 extending downwardly into the spectrophotometer 10and interrupting the beam path 12. The device 14 has a front plate 18 onwhich is positioned a power source switch 20 and a, mode of operationswitch 22.

Basically, the transport device comprises a driving mechanism, generallydesignated by the numeral 24, located in the upper portion of the deviceand a specimen carriage and carriage guide mechanism, generallydesignated by the numeral 26, located below the driving mechanism 24 andmounted on the downward extension 16. The specimen carriage itself,designated by the numeral 28, is seen in side view in-FIG. 3, in frontelevational view in FIG. 2 and in top view in FIG. 4 and includes aspecimen-containing location having a V-shaped back wall 32 andspecimen-retaining spring 34 biased toward the back wall 32. A specimentube 36, having a closed lower end and adapted to contain the specimento be analyzed, fits securely between the V-shaped rear wall 32 and thespring 34 and rests on the carriage 28 at the bottom of thespecimen-containing space 30. The specimen tube 36 is constructed ofmaterial which is of good optical quality so that it does not cause anyvariation in the beam of light in the spectrophotometer as it is movedvertically through that beam.

At the rear of the specimen carriage 28, there are provided a pair ofoutwardly or sidewardly extending guide wings 38 which are received incomplementary shaped elongated notches or ways formed in a verticalsupport member 40. The vertical support member 40 comprises a portion ofthe frame of the transport device 14 and the interaction of the wings 38on the carriage 28 and the complementary slots in the support member 40provide a smooth, reproducible vertical path of movement for thecarriage 28.

Extending downwardly from the driving mechanism 24 is an accuratelymachined lead screw 42 which is engaged within a complementary threadedaperture 44 formed vertically in the carriage 28 between the two wingportions 38. As may be best seen in FIG. 4, the support .member 40 iscut away to provide room along its length for the portion of the body ofthe carriage 28 which includes the threaded opening 44. It will bereadily appreciated that as the lead screw 42 turns in a clockwisedirection (as viewed from above) the carriage 28, and a specimen mountedtherein, will be moved vertically upwardly whereas counterclockwiserotation will produce downward vertical movement of the carriage 28.

The lead screw 42 providing the drive means for the carriage 28 ismounted by means of a bushing 46 secured on the bottom plate 48 of thedrive mechanism 24 and provide both axial and transverse support for thelead screw 42. The threaded aperture 44 of the carriage 28 aids thebushing 46 in securing the lead screw 42 against transverse movement.

As may best be seen in FIGS. 2, 3 and 4, a bracket 50 is providedextending downwardly from the plate 48 and has a pair of forwardlydirected arms 52, 54, each of which is bent toward the other andterminates at a point aligned with the beam path 12. The arms 52, 54carry appropriate lens elements 56, 58 to focus and/or align the beam oflight along the beam path 12. As seen in the view of FIG. 4, the lenses56, 58 are positioned along either side of the location of the specimentube 36. Of course, the arms 52, 54 and the lenses 56, 58 mountedtherein remain stationary during the entire operation of the device 14.An adjustment assembly 60 (see FIG. 3) is provided to adjustably mountthe bracket 50 on the bottom plate 48 so that exact alignment of thelenses 56, 58 with the beam path 12 may be easily accomplished.

Prior to a discussion of the details of the driving mechanism 24 and themicroswitches. which control the driving elements and therefore themovement of the carriage 28, reference will be made to FIG. 6 for adescription of the various movements of the carriage 28.

An access port 61 is provided in the plate 48, in alignment with thespecimen-receiving cavity 30* of the carriage 28, and when the carriage28 is in its uppermost position (the LOAD position shown in phantom inFIG. 3), the operator of the spectrophotometer may easily place thespecimen tube 36 into its appropriate spot on the carriage. In a diagramof FIG. 6, the upper edge of the specimen tube has been arbitrarilyselected as the reference point, it being understood that this is merelya diagrammatic illustration and any given point could have been chosenas a starting reference. In position A, the LOAD position, the specimentube is shown at the point it is loaded into the carriage. By operationof the driving mechanism 24, through a switch 222, the carriage 28 ismoved downwardly from its loading position to its lowermost or READYposition, labeled B in the diagram of FIG. 6. At this position, the beampath 12 is approximately aligned with the meniscus of the specimen suchthat as the tube 36 is raised upwardly slowly through the scanningmovement of the carriage 28, the beam will pass through the specimenalong the entire vertical extent of the specimen. Scanning movement,which is much slower than that of the movement from the LOAD to theREADY position, is initiated by placing the mode of operation switch 22into its SCAN position. At the completion of the scanning movement, thecarriage 28 is located at an intermediate position along its path ofvertical movement at position C in FIG. 6). At this point, the operatormay set the mode switch 22 to its READY position to move the carriage 28back to the READY position in order to prepare it for another scanningmovement; alternatively, the operator may move the mode switch 22' toits LOAD position which will bring the carriage 28 up into the LOADposition as shown in dotted lines in FIG. 3 such that the specimen maybe removed.

A pair of motors 62, 64 and a complementary pair of electromagneticclutches 66, 68 are housed within the upper chamber of the device 14 andare connected to a common shaft 70 on which is mounted a main drivinggear 72. The main driving gear 72 on the shaft 70 is connected to adriven gear 74 which is rigidly secured to an auxiliary shaft 76 whichis axially-aligned with the lead screw 42 and is mounted for rotation ona bearing 78 positioned on an intermediate shelf 80 in the drivingmechanism 24. A universal flexible joint 82 interconnects the secondaryshaft 76 with the portion of the lead screw 42 which extends above thebearing 46. Accordingly, it will be appreciated that rotation of eitherone of the motors 62, 64, when their respective clutches 66, 68 areengaged, will cause rotation of the secondary shaft 76 and hence thelead screw 42 and thereby will produce movement of the carriage 28. Theintermediate shaft 76 at its upper end drives a counter 84 by theinteraction of the bevel gears 86, 88. The counter 84 providesconvenient visual indication of the location of the carriage 28 alongits vertical path.

A set of three microswitches is mounted along the support member 40 ofthe downward extension 16 and are positioned to be engaged by thecarriage at the LOAD position, at the READY position and at the positionat the top of the scanning movement. Specifically, a ready microswitch90 is mounted at the bottom of the supporting member 40 and engages afirst inclined surface 92 (see FIG. 2) of the carriage 28 when thecarriage has moved to its lowest position. The microswitch 90 isnormally closed and thus a circuit is opened when the carriage arrivesat the READY position thereby to de-energize the driving mechanism 24. Asecond microswitch 94 is mounted intermediate the length of thesupporting member 40 and is positioned to engage a second inclinedsurface 96 on the carriage 28 when the carriage 28 has completed itsscanning movement. This second microswitch 94 is also of the normallyclosed variety and is effective to open a circuit at the end of thescanning movement to de-energize the driving means. A third or LOADposition microswitch 98 is located at the upper end of the supportingmember 40 and engages the second inclined surface 96 of the carriagemember 28 when the carriage member has been transported up to the LOADposition. This microswitch is also of the normally closed type and opensthe circuit to de-energize the driving mechanism 24 when the carriage 28has reached the LOAD position.

Reference will now be made to the schematic electrical diagram of FIG. 5for a description of the electrical circuitry employed in the drivingand switching mechanisms of the transport device 14. The power sourceswitch 20 is connected to appropriate power lines either directly (whenthe switch is set in its LOCAL position) or through thespectrophotometer (when it is set in its SPECT. position). In its OFFposition, of course, no power is sent to the unit. The SPECT." positionallows the operator to set up the transport device 14 in position tooperate as soon as an appropriate switch on the spectrophotometer isactuated. This control is schematically illustrated by the switch 100.The mode of operation switch 22 has two active arms 22A and 22B whichcontrol the mode of the motors 62, 64 and clutches 66, 68, respectively.Specifically, in the position of the various switches as shown in FIG.5, the carriage 28 is in its READY position and the switch 22 has justbeen turned to the SCAN position to energize the low speed scanningmotor 64 and the scanning clutch 68 to drive the carriage 28 through itsscanning movement. Specifically, it will be seen that a power circuitexists from the power switch through the motor control branch 22A of theswitch 22, through the motor 64 and then, by a return circuit, throughthe third microswitch 98. The scanning clutch 68 is similarly energizedthrough the clutch control branch 22B of the switch 22, through thesecond microswitch 94 to the clutch 68 and then, by a similar returncircuit, through the third microswitch 98. It will be appreciated thatas the carriage 28 moves upwardly, its second in-. clined surface 96will engage the second microswitch 94 thereby opening it and therebyde-energizing the scanning clutch 68 to halt the scanning operation.When the carriage 28 has reached this point, the operator may eitherreturn the carriage to the READY position to make another SCAN or maybring it upwardly to the LOAD position to change the specimen. In orderto repeat the scanning procedure, the operator turns the switch 22 toits READY position which, of course, deenergizes both the scanning motorand clutch circuit. It will be obvious that the motor control element ofthe switch 22A ener gizes one-half of the high speed motor 62 (its upperhalf in the schematic of FIG. 5) and the circuit through that motor iscompleted through the main branch of the switch 22. Similarly, theclutch 66 is energized through the clutch element 22B of the switch 22,through the normally closed first microswitch 90, through the solenoidof the clutch 66 and through the main elements of the switch 22. Themotor 62 is built to rotate approximately five times more rapidly thanthe motor 64 thereby to make the return movement of the carriage 28substantially faster than its scanning movement. When the carriage 28has returned to the READY position, its first inclined surface 92contacts the first microswitch thereby deenergizing the clutch 66 andhalting movement of the carriage 28. The operator may then repeat thescanning movement by moving the mode switch 22 to its SCAN position asdescribed above.

The operator may bring the specimen carriage 28 to the LOAD position(seen in dotted lines in FIG. 3) at any time by moving the mode switch22 to its LOAD position. The motor portion 22A of the mode switch 22energizes the high speed motor 62 to rotate in a direction which willmove the carriage 28' upwardly and the clutch portion 22B of the modeswitch 22 energizes the clutch 66 directly (not through the firstmicroswitch 90) and through the third microswitch 98. The carriage 28moves upwardly to the top of its path of travel until the inclinedsurface 96 contacts the microswitch 98 thereby opening this circuit anddeenergizing both the clutch 66 and the motor '62, thereby haltingmovement of the carriage 28. After a new specimen has been placed in thecarriage 28, it is returned to its READY position by moving the mode ofoperation switch 22 to the READY setting which, as stated above,energizes the high speed motor 62 in a downward direction as -well asthe clutch 66, thereby to transport the carriage 28 all the way down tothe READY position at which time the microswitch 90 is engaged, haltingfurther downward movement of thecarriage 28. The scanning movement maythen proceed in the manner described above.

From the foregoing, it will be appreciated that an investigator using anotherwise conventional recording spectrophotometer, but provided withthe specimen transport device of the present invention, may obtain agreat deal more information than was previously available to him. On theother hand the increased information can be obtained with little or nomore difliculty than was encountered in spectrophotographic examinationsaccording to the prior art. Specifically, the investigator may obtain achart or graph generally similar to that shown in FIG. 7 wherein opticaldensity is plotted on the vertical axis and location along the length ofthe specimen tube 36 is plotted along the horizontal axis. At each bandof high density, the graph will give an appropriate reading. Thus, inaddition to simply determining the various optical densities of theportions of the specimen, the investigator will have specificinformation as to the relative locations and sizes of those bands andthus have significantly more information as to each specimen than couldbe obtained from prior art investigations.

The above brief description of one presently preferred embodiment of theinvention is merely illustrative. The scanning mechanism described movesthe specimen with respect to the beam of light; it is apparent that thebeam could be moved with respect to a stationary specimen in order touse the teachings of the disclosed invention. Furthermore, although theexample given above uses a spectrophotometer as the measuring device,other devices may be used to scan and measure the opticai qualities ofthe specimen without departing from the gist of the invention. Manyother structures can be constructed to incorporate the basic teachingsof the invention departing in major or minor detail from that shown inthe drawings and described above. Accordingly, the foilowing claimsshould be construed broadly in a manner consistent with the spirit andscope of the invention.

What is claimed is:

1. An improved specimen mount and transport device for use in aspectrophotometer having a beam of light along a beam path comprising aframe,

a downwardly extending vertical support member attached to the frame forextending downwardly into said spectrophotometer a distance suflicientto interrupt the beam path thereof, said support member having elongatedways formed therein,

a specimen carriage,

means on said specimen carriage for securing a specimen in a verticalorientation at a specimen location in said beam path,

outwardly extending guide wings formed in the specimen carriage to bereceived in the elongated ways of said vertical support member, theinteraction of said wings and complementary ways providing a smooth,reproductable vertical path of movement transverse to said beam path forsaid specimen carriage,

drive means for moving said carriage upward, through a scanning movementat a scanning speed from one vertical side of the beam path to the otherside thereof, for returning the carriage to a ready or lowermostposition prior to scanning and for moving the carriage to a load oruppermost position, and

switch means for governing said drive means for effectivelyde-energizing said drive means at the completion of said scanningmovement.

2. A specimen mount and transport device as defined claim 1 wherein saidelongated ways defines a first path of scanning movement from below saidbeam path to above said beam path and a second path which is a linearextension of said first path for moving said carriage to a load positionat which a specimen may be easily loaded therein.

3. A specimen mount and transport device as defined in claim 1 whereinsaid switch means comprises microswitches mounted along said verticaisupport member in a position so as to be actuated by the specimencarriage at the ready position prior to scanning of the specimen, at theload position and at the end of said scanning movement to halt saidscanning movement.

4. A specimen mount transport device as defined in claim 1 includingindication means operatively connected to said drive means providing anindication of the position of a portion of said specimen whereby, onspectrophotometric measurement of a plurality of portions of saidspecimen, said position may be identified with said portion.

5. A specimen mount and transport device as defined in claim 1 includinga pair of parallel arms extending horizontally from said verticalsupport member and secured to said vertical support member at a pointcorresponding to the beam path of the spectrophotometer, each of thearms having an opening therethrough at a point in the path of said beamof light and lenses mounted in said openings of said arms to collimatesaid beam of light coming from the spectrophotometer.

6. An improved specimen mount transport device for use in aspectrophotometer having a beam of light along a beam path comprising aframe,

a downwardly extending vertical support member attached to the frame forextending downwardly into said spectrophotometer a distance sufficientto interrupt the beam path thereof, the support member having elongatedways formed therein,

a specimen carriage,

means on said carriage for securing a specimen in a vertical orientationin said beam path,

outwardly extending guide wings formed in the specimen carriage to bereceived in the elongated ways of said vertical support member, theinteraction of the wings and complementary ways providing a smooth,reproductable vertical path of movement transverse to said beam path forsaid specimen carriage, the beam path sweeping the vertical extent ofsaid specimen,

driving means for moving said carriage upward at a scanning speed fromone vertical side of said beam path to the other vertical side thereof,for returning the carriage to a ready or lowermost position prior toscanning and for moving the carriage to a load or uppermost position,said driving means comprising a pair of power means connected throughclutches to a common shaft, one power means providing a first and slowspeed for scanning movement and the other power means a second andfaster speed for movement of said carriage to said ready and loadpositions,

a main driving gear rigidly mounted on said common shaft,

a driven gear in mesh with said driving gear,

an auxiliary shaft to which the driven gear is rigidly secured,

a lead screw in axial alignment with said auxiliary shaft and securedthereto at its upper end, the lower end of said tead screw beingoperatively connected to said specimen carriage such that, on rotationof said screw, the1 specimen carriage is moved in a vertical direction,an

switch means for automatically governing said drive means foreffectively de-energizing said drive means at the completion of saidscanning movement.

References Cited UNITED STATES PATENTS 525,780 9/1894 Boynton 74-42483,226,556 12/1965 Rosin 3'56l05 3,320,148 5/1967 Skeggs 356-l05X3,320,149 5/1967 Isreeli 356106X 3,399,308 8/1968 Taylor 250 2352,971,431 2/1961 Glenn 8814'SI 2,982,170 5/1961 Wyss 88'-14LL 3,241,4323/1966 Skeggs et al 88l4(ZT) OTHER REFERENCES Schachman: TheUltracentrifuge: Problems and Prospects, Biochemistry, vol. 2', No. 5,Sept. 20, 1963, pp. 887-901 relied on.

RONALD L. WIBERT, Primary Examiner F L. EVANS, Assistant Examiner US.Cl. X.R.

